A High Throughput Method to Predict Skin Penetration and Screen Topical Formulations

In past decades, the understanding of mechanisms for skin penetration has significantly improved.¹ Studies have identified the stratum corneum (SC), the outermost layer of the skin, as the main barrier for penetration of pharmaceutical and cosmetic ingredients,² and due to its unique composition—i.e., about 50% ceramides, 35% free fatty acids and 15% cholesterol—the SC differs from other biological membranes.³ To facilitate the development of transdermal delivery for the compounds of interest, estimations of skin penetration rates and comparisons of dermatological formulations have become crucial to pharmaceutical and cosmetics research.⁴

Several methods have been developed for the prediction of skin penetration. Although the most useful data is obtained from in vivo studies in humans, such measurements are expensive, labor-intense and slow to perform. This makes their application in lead selection and optimization impractical. In vivo measurements using rodents⁵ are well-known to overestimate the penetration rate since compounds can permeate down the hair follicle,⁶ and although pig ear skin is also used for in vitro tests based on its structural equivalence to human skin,⁷ the most generally accepted in vitro permeation models are based on human skin as the membrane.

Static and flow-through Franz diffusion cell systems are most commonly used^{8, 9} for the implementation of these in vitro models; however, measurements based on them are only partially standardized and a broad range of experimental setup and equipment conditions can cause significant intra- and inter-laboratory variations in permeability results.^10–13 The Franz cell apparatus is also inefficient when a large number of active compounds and/or their formulations must be tested in parallel.

Considering these limitations, the authors developed a parallel artificial membrane permeability assay (PAMPA), which was introduced¹⁴ in the pharmaceutical drug discovery process as a low-cost method for high throughput measurements to predict the passive membrane permeability of molecules. This method uses a 96-well microtitre “sandwich” plate assembly to form 96, two-chamber cells used as donor/receiver compartments for permeability studies (see Figure 1).

Since the first iteration, more targeted PAMPA models have been developed, e.g., for the prediction of gastrointestinal absorption,^15–17 modeling the blood-brain barrier,¹⁸ and recently, for predicting transdermal penetration.¹⁹ The latest transdermal model of the PAMPA, the Skin PAMPA, incorporates a mixture of synthetic analogs of ceramides, cholesterol and free fatty acids in a ratio mimicking the composition of the stratum corneum lipid matrix.¹⁹ Such a skin-mimetic composition was lacking in the earlier artificial skin models.²⁰ The latest model was also optimized to correlate with highly standardized Franz diffusion cell permeability measurements through heat separated human epidermis.

The present work evaluates the ability of the Skin PAMPA¹⁹ to differentiate between topical formulations and compares the results with data obtained from in vitro Franz cell permeability measurements common in the pharmaceutical and cosmetic industries. The aim was to demonstrate the ability of this model to be easily standardized and provide high reproducibility yet remain more cost-effective and less labor-intensive than other current methods.

Materials and Methods

Chemicals^a: A novel silicone-based anhydrous gel formulation containing 5% ibuprofen (Formula A) and a viscous fluidlike test formulation containing silicone and an acrylic co-polymer (Formula B) were provided for the study^b. A commercial benchmark 5% ibuprofen formulation (Formula C) along with three available over-the-counter 1% formulations of diclofenac^c–e also were used. Universal buffer^f was adjusted to the required pH with 0.5 M NaOH, and spectroscopic- grade dimethyl sulfoxide (DMSO) also was obtained^g.

Method: A system designed for high-throughput permeability and membrane retention measurements^h was used for liquid handling, UV data collection and permeability data analysis. The 96-well PAMPA sandwiches^j used are pre-coated with a skin-mimetic artificial membrane containing a mixture of certramide, i.e., synthesized ceramidelike analog,²¹ cholesterol and free fatty acids. These sandwiches are pre-loaded with stirring disks. UV plates^k also were obtained for the study, and a stirring device^m from the company was used. Before the assay was performed, the membranes were hydrated overnight by immersion of the top (acceptor) compartment of the assembly into ionic strength pH 7.4-adjusted buffer. Hydration of the membrane ensures that it poses both hydrophobic and hydrophilic properties, which are essential to mimicking the stratum corneum barrier. Before forming the sandwich, the bottom (donor) plate was prefilled with a 180-μL aqueous solution of test compounds indomethacin, warfarin, piroxicam, niflumic acid, progesterone, carbamazepine, chlorpromazine and verapamil, dissolved in the buffer^f at various pH values and containing 0.5–1% v/v DMSO. The acceptor plate was filled with 200 μL of the buffer^f at pH 7.4. The resultant sandwich was incubated with stirring at room temperature for 5 hr.

After the permeation time, the PAMPA sandwich was separated and 150 μL of both the donor and acceptor compartments were transferred to UV plates. UV absorption (220–500 nm) was measured with a UV-Vis plate reader driven by software^h. The model compound concentrations in the buffer were chosen according to their solubility and UV detection limits (50–300 μM). The data was measured with at least six replicates on each plate, and the experiment of every plate was repeated three to five times within a six month period. Permeability values were calculated taking into account mass balance as described previously:¹⁵

Eq. 1   

where P_e is the effective permeability coefficient (cm/s); A is the filter area (0.3 cm²); V_D and V_A are the volumes in the donor (0.18 cm³) and acceptor phase (0.2 cm³); t is the incubation time (s); Τ_LAG is the steady-state time(s) estimated empirically; C_D(t) is the concentration (mol cm^-3) of the compound in the donor phase at time t; C_D(0) is the concentration (mol cm^-3) of the compound in the donor phase at time 0; R is the membrane retention factor, determined by:

Eq. 2   

and r_a is the sink asymmetry ratio (gradient-pH-induced), defined as follows:

Eq. 3    

Concentrations of test samples present in the receiver compartment were measured by comparing the UV absorbance of the compounds measured in the acceptor wells after incubation with the UV absorbances of references at known concentrations. Permeability values could be estimated using the following formula:

Eq. 4    

Formulations: As noted, this PAMPA method was modified to study topical formulations, i.e. creams, gels, ointments, etc. In this study, formulations of ibuprofen and diclofenac were used. First, hydration of the PAMPA membrane was carried out as described. Formulations were then placed in the top compartment (30 ± 5 mg per well) of the 2-chamber PAMPA sandwich while the bottom compartment was filled with 180 µL pH 7.4 buffer^f, serving as the receiver chamber (see Figure 2). Stirring in each well of the bottom (receiver) compartment was carried out by the previously mentioned device^m.The concentration of ibuprofen and diclofenac penetrating from formulations to the receiver compartment of the PAMPA sandwich was measured similarly to the method described above. Permeability values were not calculated in the case of formulations.

Franz cell measurements: In vitro Franz cell permeability experiments were conducted with the test formulations for 8 hr at 32°C. Heat-separated epidermis^22–25 from two human donors was used. The receptor fluid was phosphate buffered saline of pH 7.4. Approximately 15 mg of the test formulations were homogeneously (visually) spread over a 0.63-cm² permeation area. A commercial ibuprofen benchmark was evaluated at the same time. Triplicate cells were used for each formulation, and the ibuprofen concentration was measured using ultra-performance liquid chromatography.²⁶

The cumulative amount of drug permeated through the skin was plotted as a function of time. The steady state of diffusion can be identified by the linearity of the concentration-time curve. The flux, J, corresponds to the slope, mol hr^-1, of the curve. The permeability coefficient (K_p in cm/hr) can be calculated using the following expression:

Eq. 5    

in which A represents the actual diffusion surface area (0.63 cm²) and C₀ (mol cm^-3) corresponds to the initial concentration in the donor phase during the experiment.

Results and Discussion

Stability of pre-coated membranes: A set of pre-coated sandwich plate assemblies was stored at ambient temperature (23 ± 3°C) for a period of six months. Permeability for a set of eight test compounds, representing a broad spectrum of lipophilicity at pH 6.5, was measured after one week, one month, three months and six months following the preparation of the plates. Figure 3 shows the averaged effective permeability (P_e in 10^-6 cm/sec) and standard deviations for the set. The standard deviation for the measurements was within 0.2 logarithm permeability units, which is consistent with historic data from other PAMPA models.¹⁶ This study demonstrated that Skin PAMPA assemblies can be stored for up to six months at room temperature without compromising permeability measurements.

Topical formulations: The ability to demonstrate that this method can also be applied for comparative testing of topical formulations was one of the main goals of this work. Non-steroidal anti-inflammatory drug ibuprofen was therefore selected as a model compound for the study, as described earlier. The concentrations of ibuprofen permeating into the receiver compartment were measured at 30 min, 60 min and 150 min by means of a UV/VIS plate reader. Figure 4 compares the UV spectra for ibuprofen in the receiver compartment after having penetrated through the PAMPA barrier from the different formulations after 60 min. The spectral influence of the formulations was subtracted by using blank wells with formulations having no active pharmaceutical ingredient (API).

Even a quick visual comparison indicates that Formula A provided the highest cumulative amount of ibuprofen, compared to the other formulations or to the slurry. Quantitative analysis indicated that the amount of ibuprofen permeated from a silicon-based formulation (Formula A) was about three times the amount that permeated from the commercial benchmark at corresponding time points. These results were compared to the data for Formulas A and C measured using Franz cells and human epidermis as a barrier.²⁶ Figure 5 demonstrates that Skin PAMPA results for ranking order of Formula A and Formula C agree with the conclusions obtained from ibuprofen flux measurements through human epidermis.

Figure 6 illustrates the ability of Skin PAMPA to differentiate between three commercially available formulations of diclofenac. These examples of successful application of Skin PAMPA to study the permeability of API from topical formulations indicate that this method could also be applied to investigate the penetration of cosmetic actives from formulations.

Conclusions

The Skin PAMPA test model described was developed for the prediction of skin penetration in the early stages of drug discovery. Such a model could also be used for topical formulation testing in cosmetic research. Although the artificial membranes include no active biological elements such as skin metabolism, the system is skin-mimetic since it contains components similar to human SC, i.e., the penetration rate-limiting barriers in skin. The standardization potential and high-throughput nature of this approach can be a valuable, cost-effective complement to Franz cell permeability experiments for early skin penetration prediction, topical formulation ranking, and for understanding the effect of each formulation component on penetration phenomena.

References

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